Suppression of Streptococcosis and Modulation of the Gut Bacteriome in Nile Tilapia (Oreochromis niloticus) by the Marine Sediment Bacteria Bacillus haynesii and Advenella mimigardefordensis

ABSTRACT Streptococcosis is one of the major threats to Nile tilapia (Oreochromis niloticus) in most regions of the world. Recently, Enterococcus faecalis has been widely reported to be involved in streptococcosis in O. niloticus in Asia and Africa. This study aimed to isolate beneficial marine bacteria to evaluate their effects on growth, hematological parameters, nonspecific immunity, the gut bacteriome, and streptococcosis prevention efficacy in O. niloticus. A total of 36 marine soil bacteria were isolated, and in vitro screening was conducted to determine their antibacterial activities against fish pathogens. Two antagonistic bacteria were identified based on 16S rRNA gene sequencing, Bacillus haynesii CD223 and Advenella mimigardefordensis SM421. These bacteria were incorporated into fish feed and fed to O. niloticus for 90 days. The application of these strains via incorporation into fish feed significantly promoted growth, improved hematological parameters and immunoglobulin M (IgM) levels, modulated the gut bacteriome by reducing the load of pathogenic Enterococcus spp., and developed disease prevention efficacy in O. niloticus. Furthermore, in vivo assays revealed that the inclusion of extracellular products (ECPs) (at 250 μg mL−1) of CD223 and SM421 with feed significantly enhanced the rate of survival (100%) of O. niloticus from streptococcosis compared to the controls (only 30%). The ECPs of these bacteria also prevented 90 to 100% of fish from developing streptococcosis. These strains could be promising for safe use in O. niloticus farming to prevent and control the emergence of streptococcosis caused by E. faecalis. IMPORTANCE Nile tilapia (Oreochromis niloticus) is one of the most economically important cultured fish species throughout the world. Streptococcosis is a significant threat to global Nile tilapia farming. Enterococcus faecalis has recently emerged as an important pathogen of streptococcosis in Asia and Africa. The application of antibiotics and probiotics and vaccination are the major ways to combat streptococcosis. However, the extensive use of antibiotics leads to the development of antibiotic resistance in pathogenic as well as environmental bacteria, which is a great threat to public health. There is no study on preventing streptococcosis caused by E. faecalis using beneficial bacteria. For the first time, the present study demonstrated that two marine bacteria, Bacillus haynesii strain CD223 and Advenella mimigardefordensis strain SM421, have great potential for controlling streptococcosis in Nile tilapia. These bacteria also enhanced the growth, improved hematological parameters and IgM levels, and positively modulated the gut bacteriome of Nile tilapia.

Gram-positive Enterococcus faecalis (Ef) belongs to the lactic acid bacteria (LAB) group. Ef is formerly classified as part of the group D streptococcus system. These bacteria is found in a diverse number of environments such as the gastrointestinal tract of healthy humans. However, it is a opportunistic pathogen in humans, causing even life-threatening infections, especially in hospital environments, were naturally high levels of antibiotic resistance Ef contribute to pathogenicity. In aquaculture nutrition field, it is well known that Ef, was isolated from the intestines of healthy humans and demonstrated Immune stimulation properties and adjuvant benefits when administered in a lysed or inactivated form.
It is well known that Nile tilapia, is the most important aquacultured species in the tropical regions of the planet. Although, tilapia tolerate a wide gamma of adverse water quality and external stressors better than most commercial aquaculture species, it is evident that this species has certain susceptibility to specific pathogens. These pathogens produce a diverse number of diseases. Such as Streptococcosis, caused by Streptococcus iniae and S. galactidae or Septicemia caused by Aeromonas hydrophila or Trichodina, produced by a protozoan parasite or Columnaris caused by the myxobacteria, Flexibacter columnaris.
Although this manuscript presents results derived from a high quality research (novelty) and well designed study, there are some relevant concerns related to experimental design. Among these concerns, surge the question of why authors did select Enterococcus faecalis as pathogen for this study? If there are other more recurrent pathogens affecting Nile tilapia?. To this respect, reviewer strongly suggest that authors should include (in introduction), a brief paragraph talking about how E. faecalis infect Nile tilapia cultures in Bangladesh. I mean, how this pathogen agent is able to reach rearing water of farms, since E. faecalis is per sé originated in human intestines hence present in urban waste waters.
The other big concern is related to probiotic microorganisms used in present study. Although Bacillus haynesii strain CD223 and Advenella mimigardefordensis strain SM421 were isolated from marine soil, they were used as probiotic agents in a freshwater species. In reviewer´s opinion, probiotic -prebiotic research, should be targeted to candidate microorganisms highly available in similar environments where the host is reared.
On the other hand, it is strongly recommended all along the manuscript, Enterococcus faecalis should be mentioned only at the beginning. Subsequently, it can be mentioned as E. faecalis.
Finally, in the manuscript, M&M and discussion are misplaced. Results are misleading since in some parts, it seems that are mixed with M&M. *Note: Please, see attached manuscript file, in order to see more suggestions and comments.
Staff Comments:

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Introduction
Comment 2: Line 75: Please do not start a sentence with 'But'.

Results
Comment 3: Please avoid repetitions in the 'Results' section. I find many repetitions between 'Results' and 'Materials and methods' sections.
Comment 4: Line 145 and Line 147: I would present these results in a table. You may also add them in Table 3.
Comment 6: Lines 286-288: Same information is available in Line 145 and 147. Please avoid repetitions.
Comment 7: Lines 292-294 and Line 328: Did authors measure digestive enzyme activities in Tilapia? I do not see any results regarding this in the manuscript. Even though the isolated strains showed enzymatic activities, we can not say they improved enzyme activities in the intestine of tilapia without measuring digestive enzyme activities in tilapia.

Materials and methods
Comment 8: Line 519: Did the authors extract DNA from the digesta of hindgut? Please mention.
Comment 9: Line 560 and Line 608: Same sub-topic. Please use more specific sub-topics.
Comment 10: Line 583: The authors have used the same abbreviations (i.e., T1, T2 and T3) to denote three other diets in Line 482.

Tables
Comment 11: Please provide footnotes for abbreviations used in the tables.

Response to Reviewer
Dear Reviewer, Thank you so much for your review and specific comments and valuable suggestions to improve the manuscript. We have revised the manuscript according to your valuable comments and suggestions. Our point-by-point responses are listed below.

Reviewer #1
Reviewer's comment: Reviewer #1 (Public repository details (Required)): I recommend to deposit the raw 16S rRNA gene sequence files and metadata in a public repository.

Our response:
Thank you very much for the comment. The 16S rRNA gene sequences of these isolates CD223 and SM421 were deposited in the NCBI data bank with accession numbers MW512512.1, and OL960631.1, respectively. We provided the hyperlink of 16S rRNA gene sequences in the revised manuscript.

Reviewer's comment:
General comment: The manuscript provides novel information regarding the use of two potential probiotic marine sediment bacterial strains to prevent Streptococcosis in Tilapia.
This is a very comprehensive study, and the methodology seems very sound. The study results of significant relevance for the aquaculture industry. However, the manuscript requires some rearrangements in particular to avoid repetitions. The manuscript is also required to be checked for correct scientific English and for grammatical errors. In addition, there are some minor issues (see the comments listed below) that the authors should take into consideration.

Our response:
Thank you so much for your encouraging comments and suggestions. We have minimized the repetitions in this revision and rearranged the manuscript according to all reviewers' suggestions. The English has been checked and revised by a native speaker whereas grammar and language are improved through a professional editing service to increase the readability of the MS (Verification link: https://secure.aje.com/en/certificate/verify; Verification Code: DFB1-F83A-D791-8FED-CE9B). Additionally, the American English has been followed throughout the manuscript. Hope, you will find our response and revised MS satisfactory.

Introduction
Comment 2: Line 75: Please do not start a sentence with 'But'.

Our response:
We revised it.

Results
Comment 3: Please avoid repetitions in the 'Results' section. I find many repetitions between 'Results' and 'Materials and methods' sections.

Our response:
Thank you so much for the comment. We revised the manuscript thoroughly and minimized the redundancy in the 'Results' and 'Materials and methods' sections.

Reviewer's comment:
Comment 4: Line 145 and Line 147: I would present these results in a table. You may also add them in Table 3.

Our response:
We revised it according to the reviewer's suggestion.

Our response:
Revised.

Reviewer's comment:
Comment 6: Lines 286-288: Same information is available in Line 145 and 147. Please avoid repetitions.

Our response:
Repetitive information has been removed in the revised MS.

Reviewer's comment:
Comment 7: Lines 292-294 and Line 328: Did authors measure digestive enzyme activities in Tilapia? I do not see any results regarding this in the manuscript. Even though the isolated strains showed enzymatic activities, we can not say they improved enzyme activities in the intestine of tilapia without measuring digestive enzyme activities in tilapia.

Our response:
Thank you so much for the valuable comment. We didn't measure the digestive enzyme activities in Tilapia. We only performed the enzymatic activities of the isolated strains. Yes, we agree with your comments. Therefore, in this revision, we removed the enzymatic activities section from the manuscript. We did necessary revision regarding this throughout the MS.

Materials and methods
Comment 8: Line 519: Did the authors extract DNA from the digesta of hindgut? Please mention.

Our response:
Thanks for the comment. Yes, we extracted DNA from the digesta of hindgut. We have revised the information in the MS.

Reviewer's comment:
Comment 9: Line 560 and Line 608: Same sub-topic. Please use more specific sub-topics.

Our response:
We have added specific sub-topic.

Reviewer's comment:
Comment 10: Line 583: The authors have used the same abbreviations (i.e., T1, T2 and T3) to denote three other diets in Line 482.

Our response:
Thanks for the comments. To avoid confusion, we have revised the treatment names in growth performance (Feeding Trial) experiments (F-T1, F-T2, F-T3)

Reviewer's comment:
Tables Comment 11: Please provide footnotes for abbreviations used in the tables.

Our response:
We have provided footnotes for abbreviations used in the tables.

Reviewer's comment:
Comment 12: Table 5: Would it not be better to use a nested ANOVA to analyze the results in this table? If you considered this but decided not to, what were your reasons?

Our response:
We have used nested NOVA and presented the data in a table accordingly.
Thank you respected reviewer for giving your valuable time. Hopefully, you will find our revision satisfactory.

Reviewer's comment:
Gram-positive Enterococcus faecalis (Ef) belongs to the lactic acid bacteria (LAB) group. Ef is formerly classified as part of the group D streptococcus system. These bacteria is found in a diverse number of environments such as the gastrointestinal tract of healthy humans.
However, it is a opportunistic pathogen in humans, causing even life-threatening infections, especially in hospital environments, were naturally high levels of antibiotic resistance Ef contribute to pathogenicity. In aquaculture nutrition field, it is well known that Ef, was isolated from the intestines of healthy humans and demonstrated Immune stimulation properties and adjuvant benefits when administered in a lysed or inactivated form.
It is well known that Nile tilapia, is the most important aquacultured species in the tropical regions of the planet. Although, tilapia tolerate a wide gamma of adverse water quality and external stressors better than most commercial aquaculture species, it is evident that this species has certain susceptibility to specific pathogens. These pathogens produce a diverse number of diseases. Such as Streptococcosis, caused by Streptococcus iniae and S. galactidae or Septicemia caused by Aeromonas hydrophila or Trichodina, produced by a protozoan parasite or Columnaris caused by the myxobacteria, Flexibacter columnaris.
Although this manuscript presents results derived from a high quality research (novelty) and well designed study, there are some relevant concerns related to experimental design. Among these concerns, surge the question of why authors did select Enterococcus faecalis as pathogen for this study? If there are other more recurrent pathogens affecting Nile tilapia?.
To this respect, reviewer strongly suggest that authors should include (in introduction), a brief paragraph talking about how E. faecalis infect Nile tilapia cultures in Bangladesh. I mean, how this pathogen agent is able to reach rearing water of farms, since E. faecalis is per sé originated in human intestines hence present in urban waste waters.

Our response:
Thank you so much for making these precious comments about the bacterium, Enterococcus faecalis. Yes, it is true that in aquaculture nutrition field, many strains of E. faecalis isolated from the intestines of healthy humans as well as healthy fish have already been established as probiotic bacteria to enhance the growth and immune response of many fish species globally.
However, our research group identified E. faecalis as a pathogen of Nile tilapia with high mortality during the year 2015-2017 and published the findings in Scientific Reports 6 (Rahman et al., 2017). Our further metagenomics and in vivo studies confirmed the involvement of Enterococcus species in streptococcosis of Nile tilapia in Bangladesh . We also performed whole-genome sequencing (WGS) of several fish pathogenic E. faecalis strains isolated from streptococcosis of Nile tilapia. The WGS data of two fish pathogenic E. faecalis have been deposited in the NCBI Gene Bank under accession numbers CP046022 and JADBGH010000000. After genomic analysis, we found that these strains conserved 69 virulence factors including genes associated with gelatinase toxin and capsule producing genes for their survival. Furthermore, 39 antibiotic-resistance genes against sixteen groups of antibiotics were identified in the genome sequences of the strains. All of the strains conserved multiple antibiotic-resistance genes including macrolide-lincosamidestreptogramin (MLS) resistant genes, peptide antibiotic-resistance genes (LiaR, LiaS, LiaF, MprF, GdpD, PgsA, rpoB and rpoC), isoniazid and triclosan resistant genes, diaminopyrimidine resistant genes, aminoglycosides conferring genes, cycloserine resistance genes, and fluoroquinolones and quinolones resistance genes.
Though Streptococcus iniae, S. agalactiae, S. parauberis, and S. dysgalactiae are recognized as the prominent causative agents for streptococcosis in different geographical regions.
However, Lactococcus, Vagococcus, and Enterococcus are also isolated from the diseased fish (El-Sayed and El-Gheit, 2005;Osman et al., 2017;Mishra et al., 2018). Due to inadequate diagnostic facilities at fish farms, the majority of causative agents responsible for diseases remained uncharacterized. Recent advancements in omics technologies, especially in next generation sequencing and downstream bioinformatics analysis facilitated more precise identification and in-depth analysis of microbial communities in a given sample as well as involvement of pathogens in a disease. In more recent times, E. faecalis is reported as notable causative agents for streptococcosis Ehsan et al., 2021;Osman et al., 2017). This bacterium is posing a constant threat to fisheries and aquaculture in Asian countries such as India (Arumugam et al., 2017), Bangladesh (Rahman et al., 2017;Akter et al., 2021;Ehsan et al., 2021), Philippines (Reyes et al., 2020), Indonesia (Rizkiantino et al., 2020)  Reviewer's comment: The other big concern is related to probiotic microorganisms used in present study. Although Bacillus haynesii strain CD223 and Advenella mimigardefordensis strain SM421 were isolated from marine soil, they were used as probiotic agents in a freshwater species. In reviewer´s opinion, probiotic -prebiotic research, should be targeted to candidate microorganisms highly available in similar environments where the host is reared.

Our response:
Thank you so much for the valuable comments. Marine environment represents a promising source of new, unconventional bioactive compounds with health-promoting abilities, which can be used as food supplements in freshwater fish species Nile tilapia (Makled et al., 2017).
Even though, to the best of the author's knowledge, results from numerous applied aquaculture research trials have effectively confirmed that the use of marine bacterial species isolated from marine sediments, algae, sponge and corals can improve the farming environment and used as feed supplements in freshwater fish species (Makled et al., 2017;Makled et al., 2019;Makled et al., 2020;Hamdan et al., 2016;Soltani et al., 2019;Paul et al., 2021;Rahman et al., 2020;Paul and Rahman, 2022).
Our potential beneficial marine bacteria Bacillus haynesii strain CD223 and Advenella mimigardefordensis strain SM421 did not show any cytotoxic effects, and both of the strain can grow both in freshwater nutrient broth and marine broth. In in vivo, both of the strains developed disease resistance in tilapia and did not show any adverse effects on fish behavior and hematological parameters. Tilapia are euryhaline species, and are able to live and reproduce in a wide range of salinities. Application of these marine bacterial strains (CD223 and SM421) also enhanced the growth performances significantly and developed healthy gut